SUMMARY Breast cancer is a heterogeneous disease consisting of several clinically and molecularly distinct subtypes. Compared to other subtypes, Triple Negative Breast Cancer (TNBC), which represents 15-20% of all breast cancer, occurs at a younger age and recurs more frequently with a reduced survival rate. Currently, no targeted therapies are available for TNBC. Our short-term goal is to dissect the molecular pathways that support TNBC growth and survival. Our long-term goal is to leverage these discoveries to identify novel targets suitable for therapeutic development. We focus on how normal cells and cancer cells integrate intra- and extracellular growth and nutrient signals to sustain growth and tumorigenesis. We are most interested in how two members of the extended Myc network of transcription factors integrate growth and nutrient signals. c-Myc and MondoA function in opposition to control the aggressive growth and survival of TNBC. In this application we focus on the control of gene expression by MondoA and how metabolism shapes the MondoA-dependent transcriptome in vitro and in vivo. MondoA is the principal regulator of glucose-dependent transcription with Thioredoxin Interacting Protein (TXNIP) being its best-characterized direct and glucose-induced transcriptional target. TXNIP has pleiotropic function. Best characterized among these many functions is one in fuel choice. TXNIP is a potent suppressor of glucose uptake and aerobic glycolysis, but it can also drive the catabolism of other fuels such as branched chain amino acids and ketones in mitochondria. Thus, high TXNIP levels drive fuel oxidation in mitochondria, while restricting utilization of glucose. Conversely, low TXNIP levels tip fuel use towards glucose and away from mitochondria. We have shown that several predominant oncogenes block MondoA transcriptional activity, thereby decreasing TXNIP levels. This likely contributes to oncogene- dependent aerobic glycolysis, i.e. the Warburg Effect, which is a common feature of cancer cells. We propose to study how fuel choice controls MondoA transcriptional activity in vitro and in vivo and how signals from the tumor microenvironment dictate MondoA transcriptional activity. In Aim 1, we will determine how TXNIP, hypoxia and how the balance between glycolysis and oxidative phosphorylation controls MondoA transcriptional activity. In Aim 2, we will determine whether MondoA is required for the establishment or maintenance of tumorigenesis and whether MondoA is transcriptionally active in well perfused/oxygenated tumor regions. To examine the generality of our findings, we will determine MondoA?s role in gene expression and nutrient-sensing using other breast cancer cell lines and in conditionally reprogrammed breast cancer cells from PDX models. In Aim 3, we will use genomics approaches to discover the direct MondoA-dependent transcriptome in vivo and determine how these genes collaborate with MondoA to sustain TNBC tumorigenesis. We will also discover the cofactors required for MondoA transcriptional activity in vivo using biotin proximity labeling.